1

In Situ Hybridization Protocol

See also Simmons et al. (1989) A complete protocol for in situ hybridization of messenger RNAs in brain and
other tissues with radio-labeled single-stranded RNA probes. The Journal of Histotechnology 12:169-181.

Day 1
• Label the cRNA probes with 35S-UTP isotope as per the 'Labeling Probes' protocol.

Gel for verification of probes

• A urea/acrylamide gel is used to check that the radio-labeled cRNA probe has not been degraded and
that it is in fact labeled.
• All gel and electrophoresis apparatus must be washed w/ Alconox, rinsed w/ tap water, rinsed w/
dH2O, rinsed w/ DEPC H2O and finally rinsed with 3% H2O2. Let air dry in the hood on clean
benchcoat. Set up and check for leaks w/ DEPC H2O.
• Gilson pipets should be rinsed w/ 30% H2O2; use RNase free filter tips.
• Make up gel (use only RNase free reagents) and pour into gel apparatus using a sterile disposable
1ml pipet.
• When gel is set, transfer to the electrophoresis unit. Use 1X TBE as Running Buffer.
• Load 3 x 105 cpm/lane for each probe. Add 2 µ l RNA/DNA sample buffer (6X) to the appropriate
volume of probe and make up to 20 µ l w/ DEPC H2O.
• Pre-run gel for 15 mins and then run @ 100V until the bromophenol blue band reaches
approximately 2/3 way along gel (~75 mins).
• Place gel face down on Whatmann 3MM filter paper. Label contents of each lane on the filter paper
in pencil. Wrap gel/filter paper in Saran wrap and place with gel facing up inside one of the
cardboard X-ray exposure folders stored in the cupboard under the thermocycler. Expose gel on
Kodak XAR5 film and cover with an intensifying screen (in the dark room; intensifying screen
should have taped side facing out). Close cover on folder and wrap the whole thing in aluminum
foil.
• Place in the -70°C freezer overnight. This slows down any background radiation reactions.
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Day 2
Develop X-ray film
Develop the film in the Biochem. Dept. X-ray room on the 3rd floor, SOM (alternatively use the X-ray
room on the 4th floor, SOM, room 4643; need the entry code for the door). For the Biochem Dept
machine, it should be already on so just feed in film when ready. For the back-up machine, switch the
Power switch on; if already on, press the Ready button (will know that the developer is ready because
when the film is placed against the rollers, it will automatically feed in). Make sure that the water is on.
IN THE DARK, remove the film from the X-ray folder, bend one corner to orientate film and feed
through the machine. When done, fill out log with date and no. films developed etc.

Pre-hybridization treatment for slides

• For each wash, will require a volume of 250 ml per black box.
• Take care when placing slides into or removing slides out of solutions so as not to dislodge sections
from slides - do not agitate.
• The following rinses are done to all slides, except for the RNase control slides:
1. Rinse slides for 15 min in 4% Paraformaldehyde in PBS (make up fresh - see recipe in

Fixation solutions).
2. Rinse 3 X 5 min in PBS. Wipe boxes out with Kimwipes between rinses.

Remove RNase 3. Allow slides to air dry briefly on bench.

slides 4. Rinse in 0.1 M TEA (add 25 ml 1M TEA to 225 ml DEPC H2O) for 2 mins.
5. Acetylation: rinse slides in 625 µ l acetic anhydride dissolved in 250 ml 0.1 M TEA/per
box for 10 mins. Save some of this solution for RNase control slides.
6. 2 rinses in 2X SSC (add 25 ml 20X SSC to 225 ml DEPC H2O) for 2 mins each. Save
some of this solution for RNase control slides.
7. Dehydrate by rinsing in increasing concentrations of EtOH; do not agitate; wipe boxes
with a Kimwipe between rinses:
50% EtOH, 3 mins
70% EtOH, 3 mins
95% EtOH, 3 mins
100% EtOH, 3 mins
100% EtOH
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8. Place at back of hood on clean benchcoat with Kimwipe ‘tent’ and allow to dry for ~1 hr.

• RNase slides: Make sure these slides are kept separate from other slides and change gloves after
handling them. Place in RNase solution in a glass slide well. Incubate @ 37°C for 30 mins. Rinse
slides in PBS for 10 mins. Rinse in RNasin (60 ml PBS, 15 µ l RNasin, 60 µ l DTT) for 5 mins.
Treat as per other slides starting from Step 5.

Hybridization Reaction see pg 175

NB: The amount of 35S-cRNA depends on the cpm/µ l. These are shown in blue and will vary each
time.
• Mix probe mixtures as shown below for tPA:
mtPA probes Antisense (µ l) Sense (µ l)
35
S-cRNA (1.8 x 106 cpm/µ l) (1.2 x 106 cpm/µ l)
24 6
tRNA 500 500
1M DTT 100 100
DEPC H2O 1376 1394 To bring volume to 2.0 ml
2.0 ml 2.0 ml

• Vortex and heat @ 65°C for 5 mins.

• Add Hybridization Buffer - to determine volume required, divide the concentration of hybridization
solution desired (e.g. 5 5 x 106 cpm/ml) by the concentration of the freshly transcribed probe (e.g. for
tPA Antisense 1.8 x 106 cpm/µ l). This will give the µ l probe solution needed in each ml of
hybridization buffer. see pg 180
• Vortex and centrifuge @ 4,000 rpm for 10 mins.
• Check specific activity; count 1 µ l of final probe; should be close to 5 x 106 cpm/ml (ie. 5000 cpm).
• Put 100 µ l per slide, coverslip, seal w/ DPX.
• Hybridize overnight @ 65°C in oven.
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Day 3
Post-hybridization treatment see pg 176
• This treatment uses increasing temperature and decreasing salt content to increase the stringency of
hybridization conditions. Therefore, most of the non-specifically bound probe is washed away.
Probe RNA/mRNA duplexes are very stable under high stringency conditions.
• Washes marked with * are RADIOACTIVE.
1. Soak off coverslips in 4X SSC.
*
2. 4 X 5 mins in 4X SSC.
*
RNase digestion 1 hr @ 37°C (use recipe for ~200 ml of digestion solution).
* 3.

4. Rinse & desalt - add 200 µ l 1M DTT (1mM) per 200 ml solution.
2X SSC - 5 mins
2X SSC - 5 mins
1X SSC - 10 mins To remove non-
0.5X SSC - 10 mins specific binding

* 0.1X SSC - 45 mins @ 65°C

* 0.1X SSC - 45 mins @ 65°C

NB: The final
0.1X SSC - rinse to cool slides concentration of salt and
temperature is critical.
5. Dehydrate - add 200 µ l DTT and 800 µ l 20X SCC per 200 ml.
50% EtOH - 3 mins
70% EtOH - 3 mins
95% EtOH - 3 mins
3X 100% EtOH - 3 mins each
6. Air dry 1 hr and expose to film.

Day 7
Film development
• 4 mins in Kodak D-19 solution.
• 30 secs in dH2O
• 10 mins in Kodak film-strength rapid fixer.
• 20 min rinse in running tap water.
• Air dry.
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Solutions
To maintain all solutions as RNase free, use baked (180°C) glassware, magnetic stirrers, spatulas and bottles; use
DEPC H2O; pH using sterile plastic disposable pipets and pH papers. Sterile filter using bottle-top filter or
autoclave all solutions before storing in RNase-free cabinet. Use only RNase free chemicals (containers should
be labeled). Make sure to use Molecular Biology Grade chemicals only (esp. EDTA). Label all bottles using red
autoclave tape and mark with ‘RNase Free’. For DEPC H2O and 3% H2O2 recipes, see in situ slide prep protocol.

TBE (Tris-borate) Buffer (5X)

54 g Tris (Tizma) buffer
27.5 g Boric acid
20 ml 0.5M EDTA (pH 8.0)
up to 1.0 L w/ DEPC H2O

or

54 g 27 g 13.5 g Tris (Trizma) Buffer

27.5 g 13.75 g 6.875 g Boric Acid
3.8 g 1.9 g 0.95 g EDTA (preservative; ethylenediaminetetraacetic)
up to 1.0 L up to 500 ml up to 250 ml w/ Barnstead H2O
Check regularly as tends to precipitate out. Store at room temp.

5M NaCl (FW = 58.44)

29.22 g NaCl
up to 100 ml w/ DEPC H2O
This will need to be autoclaved, will not go through bottle-top filter.

10X PBS
80.0 g NaCl
4.0 g KCl
1.5 g Na2HPO4
1.5 g KH2PO4
up to 1.0 L w/ DEPC H2O
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4% pH2CO (Paraformaldehyde) pH 8.0

Make up in the hood – toxic; needs to be made up fresh each time.
10 g Paraformaldehyde
100 ml DEPC H2O
Heat to 58°C. Add a few drops (~12-15) of 10M NaOH to clear and check pH is 7.5-8.0.
25 ml 10X PBS
Make up to 250 ml w/ DEPC H2O.
Refrigerate to bring to room temp. quickly.

0.5M EDTA (Ethylenediaminetetraacetic acid – disodium salt dihydrate) pH 8.0 (FW = 372.2)
37.22 g EDTA
100 ml DEPC H2O
Adjust pH to 8.0 using 10M NaOH (should need ~13 ml)
up to 200 ml w/ DEPC H2O

1M TEA (Triethanolamine hydrochloride) pH 8.0 (FW = 185.7)

37.14 g TEA
100 ml DEPC H2O
Adjust pH to 8.0 using 10M NaOH (should need ~6 ml)
up to 200 ml w/ DEPC H2O

20X SSC (Sodium chloride/sodium citrate) pH 7.0

SSC is similar to physiologic saline & adjusts the pH & osmolarity of tissue to normal values.
175.3 g NaCl
88.2 g Na citrate
800 ml DEPC H2O
Adjust pH to 7.0 using concentrated HCl (should only need a few drops)
up to 1 L w/ DEPC H2O
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1M Tris-Cl pH 8.0
12.1 g Tris (Trizma)
80 ml DEPC H2O
Adjust pH to 8.0 using concentrated HCl (should need about 5 ml)
up to 100 ml w/ DEPC H2O

RNase Digestion Solution See p 176 from Simmons et al. for RNase A MEB
100 µ l RNase A (10 mg/ml in dH2O, aliquoted) 120 µ l 400 µ l
5 ml 5M NaCl 6 ml 20 ml
0.5 ml 1M Tris (pH 8.0) 580 µ l 2 ml
100 µ l 0.5M EDTA (pH 8.0) 120 µ l 400 µ l
44 ml dH2O (add last to mix reagents) 54 ml 178 ml
~60 ml ~200 ml

1M DTT (Dithiothreitol) (MW = 154.25)

5g DTT (whole bottle)
up to 32.4 ml w/ DEPC H2O
Make up in hood; filter sterilize using 0.2 µ m filter attached to a 10 ml syringe.
Aliquot and store @-20°C.
Dissolved in 10mM Na acetate buffer (pH 5.2).

RNA/DNA Sample Buffer (6X)

0.025 g bromophenol blue (0.25%)
0.025 g xylene cyanol FF (0.25%)
1.5 g Ficoll (Type 400, Pharmacia) (15%)
up to 10 ml w/ dH2O
Store @ room temp.
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Hybridization Buffer
25 ml Formamide
10 ml 50% Dextran SO4 (Fridge 5)
3 ml 5M NaCl
0.8 ml Denhardt’s (50X)
0.5 ml 1M TrisCl (pH 8.0)
0.1 ml 0.5M EDTA (pH 8.0)
0.6 ml DEPC H2O
40 ml
Vortex to mix well. Make up in a sterile 50 ml centrifuge tube.
• This buffer is stable for ~1 month @ 4°C if sterility maintained.
• Formamide lowers the effective melting temp. of nucleic acid duplex chains so that hybridization can occur
at temps which preserve tissue morphology. It should be aliquoted & stored @ -20°C. If yellow, discard.
• Dextran sulfate increases the effective probe concentration @ the tissue surface. It must be very pure. Add
50 g Dextran sulfate to 70 ml DEPC H2O. Cap & dissolve for 3-4 hrs @ 68°C (waterbath). After solution
cools to room temp, increase to final volume of 100 ml w/ DEPC H2O & mix well. Store @ 4°C.
• Denhardt's is a mix of proteins which stabilizes probe and decreases background hybridization. It is quite
stable if sterility is maintained. For 50X, use 1 g Ficoll, 1 g polyvinylpyrrolidone, 1 g BSA Fraction V and
make up to a total of 100 ml with DEPC H2O. Filter sterilize, aliquot & store @ -20°C.

Urea/Acrylamide Gel
1.67 ml 30% acrylamide (5%)
5g urea (53%)
1 ml 5X TBE
6.77 ml DEPC H2O
Add last:
100 µ l 10% APS in DEPC H2O
5 µ l TEMED